We will continue studies on oncogenic transformation by jaagsiekte sheep retrovirus (JSRV), the cause of a transmissible lung cancer, ovine pulmonary carcinoma (OPA) that closely resembles human lung adenocarcinoma. The target cells for oncogenesis are type II pneumocytes and Clara cells. JSRV is unique in that its envelope gene also is an oncogene;it can transform cells in culture and induce tumors in animals. Like other viral onco-proteins, multiple domains of Env are involved in transformation. The cytoplasmic tail of the transmembrane protein (TM) is essential, but the surface protein (SU) also participates. In addition, JSRV encodes a regulatory activity rej, necessary for expression of proteins from unspliced viral mRNA. Rej is encoded within the env region, which raises the possibility that it may also be involved in transformation. The proposed research has three aims: 1) Structure-function analysis of the Env protein TM, and of rej. The structure of the cytoplasmic tail of TM will be determined by solution NMR, the potential roles of the membrane-spanning and ectodomains of TM will be investigated, and a potential role for rej in transformation will be evaluated. 2) Identification of cellular proteins involved in transformation. Candidate proteins identified by yeast two-hybrid screening will be tested for relevance to JSRV transformation, and additional cellular binding proteins will be sought by tandem affinity purification (TAP)/proteomic approaches. 3) JSRV transformation in vitro and in vivo. To more closely mirror in vivo oncogenesis of lung epithelial cells, in vitro transformation of primary rat and ovine type II pneumocytes (with or without cooperating oncogenes), and of murine bronchiole-alveolar stem cells (BASCs) will also be tested. A transgenic mouse model for JSRV-induced lung cancer will be developed, and wild-type and mutant JSRV Env's will be tested for tumorigenicity in an adeno-associated virus (AAV) vector. These experiments will substantially increase our understanding of this unique model for human lung cancer, and the oncogenic pathways uncovered may elucidate similar ones in human lung cancer. Lay summary: This proposal will study a virus of sheep (JSRV) that causes a transmissible lung cancer (OPA), very similar to one form of human lung cancer. The discoveries may lead to a better understanding of human lung cancer, which may result in improved therapies and prevention.